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Sample GSM4905388 Query DataSets for GSM4905388
Status Public on Jul 26, 2022
Title H2B8_seedling_input_rep1
Sample type SRA
Source name 35Sp::H2B.8-eGFP seedling
Organism Arabidopsis thaliana
Characteristics ecotype: Col-0
genotype: 35Sp::H2B.8-eGFP
cell type: ten-day-old seedlings
chip antibody: none
Growth protocol Plants were grown under long day (16 hr light; 8 hr dark) conditions at 22 °C.
Extracted molecule genomic DNA
Extraction protocol Ten-day-old seedlings of a 35Sp::H2B.8-eGFP line were ground with a pestle and mortar in liquid N2 and homogenized in nuclei isolation buffer (0.25 M sucrose, 15 mM PIPES pH 6.8, 5 mM MgCl2, 60 mM KCl, 15 mM NaCl, 1 mM CaCl2, 0.9% Triton X-100, 1 mM PMSF, 1x proteinase inhibitor Cocktail). Nuclei were separated from debris by filtering through two layers of miracloth. Chromatin was fragmented by micrococcal nuclease (MNase, New England Biolabs) digestion. For IP samples, fragmented chromatin was immunoprecipitated with GFP-Trap beads. For input, no immunoprecipitation was undertaken. Protein and RNA was removed by digestion with Proteinase K and RNase A, respectively. Phenol-chloroform extraction was used to obtain DNA.
Libraries were prepared using the Ovation Ultralow System V2 (Nugen, 0344) and sequenced on the NextSeq 500 (Illumina) with 2 × 38 bp paired end reads at the John Innes Centre, Norwich, UK.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
Description H2B8_seedling_peaks.bed
Data processing ChIP-seq: Reads were mapped to TAIR10 using Bowtie2-, retaining mononucleosomal fragments with using parameters -I 130 -X 200. Mapped reads were sorted and indexed using SAMtools-1.9.
ChIP-seq: Bigwig files were generated by normalising IP bam files to respective inputs with the bamCompare tool from deepTools-3.1.1. Bigwig files of enrichment were generated at single base resolution (w1) and at 50 bp intervals (w50).
ChIP-seq: To generate peaks, H2B.8 enrichment was calculated over 50 bp windows and those with > 0.5 log2(IP/input) were retained. Windows within 501 bp were merged using BEDtools-2.28.0. Regions were filtered by size, with those < 201 bp removed from analysis. H2B.8 enrichment was then calculated over the new regions, those with < 0.5 log2(IP/input) were discarded. The remaining regions were defined as H2B.8 peaks.
ChIP-seq: Overlaps with genes and TEs were determined using BEDtools-2.28.0; 50% of the feature was required to be overlapped by a peak to be defined as an overlap.
Genome_build: TAIR10
Supplementary_files_format_and_content: ChIP-seq: Bigwig files generated with bamCompare in deepTools-3.1.1. Scores at w1 or w50 resolution represent log2(IP/input).
Supplementary_files_format_and_content: ChIP-seq: BED file of H2B.8 peaks in sperm and seedling.
Supplementary_files_format_and_content: ChIP-seq: BED file of genes or TEs marked by H2B.8 in sperm and seedling.
Submission date Nov 12, 2020
Last update date Jul 26, 2022
Contact name Xiaoqi Feng
Organization name John Innes Centre
Department Cell and Developmental Biology
Lab Xiaoqi Feng
Street address Norwich Research Park
City Norwich
State/province Norfolk
ZIP/Postal code NR4 7UH
Country United Kingdom
Platform ID GPL19580
Series (1)
GSE161366 Histone H2B.8 compacts flowering plant sperm through chromatin phase separation
BioSample SAMN16784504
SRA SRX9495622

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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